Tyrosine phosphorylation in peripheral T cells of kidney transplant recipients: analyses of baseline levels and response to T cell receptor stimulation.

Impaired immunosurveillance in recipients of organ transplants has been attributed to alleviation of T cell functions. We analyzed the phosphorylation of tyrosine residues in T cells of peripheral blood, and after T cell receptor (TCR) stimulation. The TCR was stimulated by OKT3 monoclonal antibody (mAb) in non-separated heparinized blood specimens of patients (n=64) and healthy controls (n=25). After fixation and red cell lysis, lymphocytes were permeabilized by saponin. Subsequently, intracellular phosphotyrosine residues and surface CD3 antigen were stained simultaneously with specific mAbs. We analyzed transplant recipients and healthy donors for baseline levels of total cellular tyrosine phosphorylation and for increase in phosphotyrosine content following stimulation by OKT3. Phosphotyrosine levels were significantly lower in non-stimulated T cells of kidney transplant recipients compared to controls (p=0.004). There was a marked variability in the levels of tyrosine phosphorylation among transplanted patients (p=0.02). T cell receptor stimulation by OKT3 mAb in vitro led to a strong increase of tyrosine phosphorylation in all specimens of patients and healthy controls. In conclusion, we demonstrated decreased phosphotyrosine levels in T cells of kidney transplant recipients compared to healthy donors. However, increase in tyrosine phosphorylation was not impaired in all patients as a result of TCR stimulation.

Flow cytometric analysis of protein phosphorylation in the hematopoetic system.

Cellular growth and differentiation in blood cells are regulated by the phosphorylation status of growth factor receptors and downstream proteins. Protein kinases and phosphatases balance the homeostasis of protein phosphorylation. Various diseases are associated with alterations in these tightly regulated processes. Aberrations have been proved to be of diagnostic value and might enhance the pathophysiological insight into the origin of the disease. However, quantitation of protein phosphorylation is currently not feasible in a clinical situation. We developed a flow cytometric methodology which enables for direct investigation of protein phosphorylation in cell populations defined by multi-color flow cytometry. This assay does not only overcome drawbacks of traditional methodologies (e.g. Western blotting) but also allows quantitative analyses even in rare cell populations. We accurately examined phosphorylation levels in different cell populations of hematological interest and especially analyzed CD34+ hematopoietic progenitor cells. CD34+ cells in bone marrow and in cord blood contained similar, low levels of phosphotyrosine. Circulating pheripheral blood system cells PBSC in patients exposed to G-CSF for stem cell mobilization exhibited significantly increased levels of phosphotyrosine. In vitro exposure of CD34+ progenitors to growth factors (G-CSF, IL-3, SCF) raised the levels of tyrosine phosphorylation in bone marrow and cord blood. Effects were dose and time dependent. Interestingly, in vivo stimulated CD34+ PBSC could not be further stimulated in vitro. In conclusion, we present a new powerful methodology for analysis of protein phosphorylation in hematological specimens. The method does not only allow for accurate detection of phosphorylation levels in vivo, but also enables for quantitative analysis of growth factor receptor stimulation in vitro and in vivo.

Alterations of protein tyrosine phosphorylation in T cells of immunocompromised patients.

Activation of the T-cell receptor (TCR) results in recruitment of tyrosine kinases and changes of tyrosine phosphorylation levels. We quantitatively analyzed protein phosphorylation of resting and TCR stimulated T cells for healthy donors and immunocompromised patients using two-colour flow cytometry. Stimulation of T cells of healthy persons by OKT3 antibody led to a biphasic increase of phosphotyrosine levels with the first peak after 15 s and the absolute maximum occurring after 3-5 min. Levels remained high up to 30 min and returned to baseline levels afterwards. Compared to healthy blood donors, the phosphotyrosine baseline levels were 20-30% increased in patients after bone marrow transplantation (BMT). Using OKT3 to stimulate T cells of BMT patients led to strong increases in phosphotyrosine levels comparable to those of controls. In contrast, the response of T cells of human immunodeficiency virus-infected patients with acquired immune deficiency syndrome was severely impaired (P = 0.01). In conclusion, this flow cytometric methodology enables analyses of changes in cellular phosphotyrosine levels following TCR stimulation. The increased baseline levels in BMT patients and the observed unresponsiveness of T cells in AIDS patients could be of interest for the study of predictors of graft-versus-host reactivity and the clinical analysis of immune functions in AIDS patients, respectively.